PROJECT SUMMARY/ABSTRACT Necroptosis (programmed necrosis) is a newly defined cell death pathway that contributes to the pathology of vascular diseases like atherosclerosis and abdominal aortic aneurysms. However, the causes of endothelial cell necroptosis and its impact on vascular integrity are unknown. The applicant's lab recently generated mice with vascular deletion of the chromatin-remodeling enzymes CHD4 or BRG1 and found that these mutants die at midgestation from endothelial cell necroptosis and vascular rupture. Importantly, transcripts of the necroptosis executor gene Ripk3 were upregulated in Chd4 and Brg1 mutant endothelial cells, and genetic reduction of Ripk3 rescued both mutants from vascular rupture. The objective of this proposal is to exploit these vascular Chd4 and Brg1 mutants to define factors that promote embryonic endothelial cell Ripk3 transcription and necroptosis. The central hypothesis is that CHD4 and BRG1 respectively maintain embryonic blood vessel and lymphatic integrity by indirectly repressing Ripk3 transcripts and endothelial cell necroptosis. This hypothesis is based on the genetic data described above and on the applicant's preliminary evidence that CHD4 and BRG1 indirectly regulate Ripk3 transcription in cultured endothelial cells. Two specific aims will be employed to test the central hypothesis and to identify factors that directly trigger endothelial cell necroptosis. First, the applicant will determine how CHD4 regulates endothelial cell necroptosis in embryonic blood vessels. Cellular, molecular, biochemical, and genetic approaches will be used to test the working hypothesis that plasmin?which is aberrantly elevated in Chd4 mutant embryos?directly triggers Ripk3 transcription and necroptosis in blood vessel endothelial cells. Secondly, the applicant will determine how BRG1 regulates endothelial cell necroptosis in embryonic lymphatic vessels. Cellular and molecular approaches will be used to test the working hypothesis that BRG1 transcriptionally regulates the Ripk3 mRNA-destabilizing complex CCR4-NOT in lymphatic endothelial cells. In addition, Brg1 mutants will be used to identify the lymphatic endothelial cells most susceptible to RIPK3-mediated necroptosis during embryonic development. The proposed research is significant because it will define mechanistic causes and effects of endothelial cell necroptosis for the first time and will fundamentally advance the understanding of how this cell death program influences developmental vascular biology. The results of this work will also set the groundwork for elucidating how endothelial cell necroptosis impacts postnatal pathologies associated with cell death and vascular fragility.